RodZ and PgsA Play Intertwined Roles in Membrane Homeostasis of Bacillus subtilis and Resistance to Weak Organic Acid Stress.

Johan Van Beilen,Christoph J Blohmke,Hendrik Folkerts,Anna Zakrzewska,Wim Kulik,Fred M Vaz,Stanley Brul,Alexander Ter Beek,Richard De Boer

doi:10.3389/fmicb.2016.01633

Johan Van Beilen, Christoph J Blohmke + Show 7 more

Open Access

https://doi.org/10.3389/fmicb.2016.01633

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Abstract

Weak organic acids like sorbic and acetic acid are widely used to prevent growth of spoilage organisms such as Bacilli. To identify genes involved in weak acid stress tolerance we screened a transposon mutant library of Bacillus subtilis for sorbic acid sensitivity. Mutants of the rodZ (ymfM) gene were found to be hypersensitive to the lipophilic weak organic acid. RodZ is involved in determining the cell’s rod-shape and believed to interact with the bacterial actin-like MreB cytoskeleton. Since rodZ lies upstream in the genome of the essential gene pgsA (phosphatidylglycerol phosphate synthase) we hypothesized that expression of the latter might also be affected in rodZ mutants and hence contribute to the phenotype observed. We show that both genes are co-transcribed and that both the rodZ::mini-Tn10 mutant and a conditional pgsA mutant, under conditions of minimal pgsA expression, were sensitive to sorbic and acetic acid. Both strains displayed a severely altered membrane composition. Compared to the wild-type strain, phosphatidylglycerol and cardiolipin levels were lowered and the average acyl chain length was elongated. Induction of rodZ expression from a plasmid in our transposon mutant led to no recovery of weak acid susceptibility comparable to wild-type levels. However, pgsA overexpression in the same mutant partly restored sorbic acid susceptibility and fully restored acetic acid sensitivity. A construct containing both rodZ and pgsA as on the genome led to some restored growth as well. We propose that RodZ and PgsA play intertwined roles in membrane homeostasis and tolerance to weak organic acid stress.

Highlights

Weak organic acids are commonly used preservatives in the food industry since they inhibit the growth of spoilage bacteria, yeasts, and molds (Brul and Coote, 1999; Davidson and Harrison, 2002; Beales, 2004; Brul and Ter Beek, 2010)
E. coli strains XL1-Blue and MC1061 were grown in lysogeny broth (LB) at 37◦C
In order to have a direct measure of functional importance for B. subtilis weak organic acid stress resistance we decided to opt for the construction and screening of a transposon mutant library for sorbic acid hypersensitive mutants

Summary

Introduction

Weak organic acids (e.g., sorbic-, acetic-, and benzoic- acid) are commonly used preservatives in the food industry since they inhibit the growth of spoilage bacteria, yeasts, and molds (Brul and Coote, 1999; Davidson and Harrison, 2002; Beales, 2004; Brul and Ter Beek, 2010). Depending on the lipophilic nature of the compound, the neutral undissociated form of the molecule is able to dissolve in and Frontiers in Microbiology | www.frontiersin.org van Beilen et al. RodZ PgsA Weak Acid Stress diffuse over the membrane. The hydrophobic tail of, e.g., lipophilic sorbic acid, can more permanently insert into the membrane perturbing its structure and interfering with the function of proteins (Sheu and Freese, 1972; Stratford and Anslow, 1998; Chu et al, 2009). The proton gradient dissipates and, depending on the buffering capacity of the cell, the cytosol may acidify, affecting oxidative phosphorylation, the transport of nutrients, and a number of other metabolic functions (Bauer et al, 2003; Cotter and Hill, 2003; Brul and Ter Beek, 2010; van Beilen et al, 2014). It has been shown that the accumulation of the anion in the cell can cause a rise in osmolarity and affect cytosolic enzymes (Azukas et al, 1961; York and Vaughn, 1964; Russell, 1992)

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